Zeitschrift für Bioverarbeitung und Biotechnik

Structure and Catalytic Mechanism of a Glycoside Hydrolase Family-127 β-L-Arabinofuranosidase (HypBA1)

Abstract

Chun-Hsiang Huang, Zhen Zhu, Ya-Shan Cheng, Hsiu-Chien Chan, Tzu-Ping Ko, Chun-Chi Chen, Iren Wang, Meng-Ru Ho, Shang-Te Danny Hsu, Yi-Fang Zeng, Yu-Ning Huang, Je-Ruei Liu, Rey-Ting Guo

The β-L-arabinofuranosidase from Bifidobacterium longum JCM 1217 (HypBA1), a DUF1680 family member, was recently characterized and classified to the glycoside hydrolase family 127 (GH127) by CAZy. The HypBA1 exerts exo-glycosidase activity to hydrolyze β-1,2-linked arabinofuranose disaccharides from non-reducing end into individual L-arabinoses. In this study, the crystal structures of HypBA1 and its complex with L-arabinose and Zn²⁺ ion were determined at 2.23-2.78 Å resolution. HypBA1 consists of three domains, denoted N-, S- and C-domain. The N-domain (residues 1-5 and 434-538) and C-domain (residues 539-658) adopt β-jellyroll architectures, and the S-domain (residues 6-433) adopts an (α/α)6-barrel fold. HypBA1 utilizes the S- and C-domain to form a functional dimer. The complex structure suggests that the catalytic core lies in the S-domain where Cys417 and Glu322 serve as nucleophile and general acid/base, respectively, to cleave the glycosidic bonds via a retaining mechanism. The enzyme contains a restricted carbohydrate-binding cleft, which accommodates shorter arabino oligosaccharides exclusively. In addition to the complex crystal structures, we have one more interesting crystal which contains the apo HypBA1 structure without Zn²⁺ ion. In this structure, the Cys417-containing loop is shifted away due to the disappearance of all coordinate bonds in the absence of Zn²⁺ ion. Cys417 is thus diverted from the attack position, and probably is also protonated, disabling its role as the nucleophile. Therefore, Zn²⁺ ion is indeed involved in the catalytic reaction through maintaining the proper configuration of active site. Thus the unique catalytic mechanism of GH127 enzymes is now well elucidated.